Purification and characterization of the plasminogen activator secreted by human melanoma cells in culture.
The plasminogen activator secreted by a cultured human melanoma cell line was purified and compared with urokinase and with tissue plasminogen activator from human uterus. The purification procedure consisted of chromatography on zinc chelate-agarose, concanavalin A-agarose, and Sephadex G-150 in the presence of 0.01% (v/v) Tween 80. The purified material was obtained from the culture medium with a yield of 46% and a purification factor of 263. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one main band with a molecular weight of about 72,000, and in the presence of reducing agents, two bands of 33,000 and 39,000. Addition of the protease inhibitor Aprotinin to the culture media and column buffers yielded a one-chain plasminogen activator with a molecular weight of about 72,000. One molecule of activator reacted with about one molecular of [3H]diisopropylfluorophosphate. The melanoma plasminogen activator and the uterine tissue plasminogen activator appeared to be very similar on dodecyl sulfate-polyacrylamide gel electrophoresis, amino acid analysis, and amidolytic properties. Both activators bound to fibrin clots, while urokinase did not. In immunodiffusion, as well as in quenching experiments of the fibrinolytic activities, the melanoma plasminogen activator appeared to be immunologically identical with the uterine tissue plasminogen activator, but unrelated to urokinase. All these findings indicate that the plasminogen activator secreted by human melanoma cells in culture is very similar to, or identical with, the plasminogen activator found in normal tissue, but different from urokinase.
D000596:Amino Acids / Q000032:analysis; D002460:Cell Line; D005260:Female; D005342:Fibrinolysis; D006801:Humans; D007106:Immune Sera; D005779:Immunodiffusion; D007700:Kinetics; D008545:Melanoma / Q000378:metabolism*; D010960:Plasminogen Activators / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D014599:Uterus / Q000032:analysis
D000596:Amino Acids; D007106:Immune Sera; D010960:Plasminogen Activators